Multi-layer microfluidic devices

ABSTRACT

The present invention provides multi-layer microfluidic systems, by providing additional substrate layers, e.g., third, fourth, fifth and more substrate layers, mated with the typically described first and second layers. Microfabricated elements, e.g., grooves, wells and the like, are manufactured into the surfaces between the various substrate layers. These microfabricated elements define the various microfluidic aspects or structures of the overall device, e.g., channels, chambers and the like. In preferred aspects, a separate microscale channel network is provided between each of the substrate layers.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation of U.S. patent applicationSer. No. 09/679,479, filed Oct. 4, 2000, which is a continuation of U.S.patent application Ser. No. 09/231,209, filed Jan. 14, 1999, whichclaims priority from U.S. Provisional Patent Application No. 60/072,001,filed Jan. 20, 1998, each of which is hereby incorporated herein byreference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

[0002] Microfluidic devices and systems have been gaining increasinginterest for their ability to provide improved methods of performingchemical, biochemical and biological analysis and synthesis. Inparticular, the small size and automatability of these microfluidicsystems provides a variety of advantages in terms of low reagentrequirements, low space requirements, shorter reaction times, andintegratability. All of these advantages together, provide systems whichcan be extremely useful in performing large numbers of reactions inparallel, in order to provide enhanced analytical throughput.

[0003] In general, the production of microfluidic devices has beenenabled by advancements in microfabrication technology used in theelectronics and semiconductor manufacturing industries. Specifically,technologies, such as photolithography, wet chemical etching, injectionmolding of plastics, and the like, have been used to fabricatemicroscale channels and wells in the surface of planar substrates.Overlaying a second planar substrate on the surface of the first createsthe microfluidic channels and chambers of the device. While thesemicrofabrication techniques permit the incorporation of relativelycomplex channel networks in a relatively small area, the ability tofurther reduce the size of microfluidic devices produced in this mannerhas been somewhat limited by the two dimensional orientation of thechannel networks. Specifically, because channel networks have beengenerally defined in two dimensions, e.g., in a single layer, differentchannel networks could not cross or otherwise occupy the same area onthe substrate.

[0004] In order to allow further reduction of microfluidic device size,it would therefore be desirable to provide microfluidic devices that arenot limited by the two-dimensional nature of typical microfluidicdevices. The present invention meets these and other needs.

SUMMARY OF THE INVENTION

[0005] The present invention generally overcomes the limits oftwo-dimensional microfluidic systems by providing multi-layermicrofluidic devices and systems (e.g., 3-dimensional).

[0006] In one aspect, the present invention provides microfluidicdevices which comprise a body structure having at least first, secondand third substrate layers, the second substrate layer disposed on topof the first substrate layer and the third substrate layer disposed ontop of the second substrate layer. The devices include at least first,second and third ports disposed in the body structure. The devices ofthe invention also include at least first and second microscale channelnetworks. The first channel network is typically disposed between thefirst and second substrate layers, and is in fluid communication withthe first and second ports, but not the third port. The second channelnetwork, on the other hand, is disposed between the second and thirdlayers, and is in fluid communication with the first and third ports,but not the second port.

[0007] The present invention also includes systems incorporating thesemicrofluidic devices, which systems typically include a materialtransport system operably linked to each of the first, second and thirdports of the device, for selectively controlling movement of material inthe first and second channel networks.

[0008] In a related aspect, the present invention provides amicrofluidic device comprises at least three substrate layers and atleast two channel networks, as described above. However, in this aspect,the device includes at least one fluid passage disposed through thesecond substrate layer, which provides fluid communication between thefirst channel network and the second channel network.

[0009] In another aspect, the present invention provides methods ofperforming a plurality of different analyses on a single fluid sample.The methods comprise providing a microfluidic device according to theinvention, e.g., as described above. The fluid sample to be analyzed isplaced into the first, common port of the device. The sample is thentransported through each of the first and second channel networks,whereby a different reaction or analysis is performed on the sample ineach of the channel networks. The result of these different reactions oranalyses are then detected.

[0010] In a more specific embodiment, the multi-layer devices of theinvention are useful in methods of sequencing nucleic acids. Inparticular, the devices for this application typically include at leastfive substrate layers. The devices also include at least four separationchannels, each of the separation channels disposed between two differentsubstrate layers. A common sample reservoir is included, connected toeach of the four separation channels via four separate sampleintroduction channels. Four reagent channels are also provided, whereineach reagent channel independently connects each of the four sampleintroduction channels with a separate one of four separate reagentreservoirs disposed in the body structure. Each of the four separatereagent reservoirs comprise a different reagent mixture of nucleotidetriphosphates, primer sequences, nucleic acid polymerases, and aseparate dideoxynucleotide.

[0011] In a related aspect, the present invention provides methods ofsequencing nucleic acids using the devices described above.Specifically, the target nucleic acid is separately combined with eachof the four different reagent mixtures in each of the four sampleintroduction channels. The products of the reaction resulting from thiscombination are then injected into the separation channel to size thedifferent products. Based upon the size of the reaction products, onecan determine the sequence of nucleotides in the target nucleic acidsequence.

BRIEF DESCRIPTION OF THE FIGURES

[0012]FIG. 1 illustrates an embodiment of a multi-layer device.

[0013]FIG. 1A illustrates the multi-layer construction of the devicefrom a perspective view.

[0014]FIG. 1B illustrates a perspective view of an assembled multi-layerdevice.

[0015]FIG. 1C illustrates a side view of a multi-layered microfluidicdevice.

[0016]FIG. 2 illustrates an embodiment of a microfluidic deviceincorporating an alternate detection window structure.

[0017]FIG. 3 illustrates an alternate embodiment of a multi-layermicrofluidic device incorporating cross-over channels.

[0018]FIG. 4 illustrates a five-layer, four channel network device forcarrying out four separate analyses on the same sample materialsimultaneously, e.g., nucleic acid sequencing.

DETAILED DESCRIPTION OF THE INVENTION

[0019] In most instances, microfluidic devices have been manufacturedusing microfabrication methods commonly employed in the electronicsindustry. Such methods generally involve the fabrication of microscalestructures, e.g., grooves, wells, depressions and the like, on the upperplanar surface of a first solid substrate material. A second substratelayer having a lower planar surface is then bonded over this surface,which covers and seals the grooves and wells to form the channels andchambers. As a result of these manufacturing techniques, microfluidicdevices most often employ a planar structure where, aside from theirintrinsic depth, the fluidic elements generally exist in two dimensions.The present invention, on the other hand, provides three dimensionalmicrofluidic devices, e.g., employing multi-layered channel structuresand networks. By providing multiple layers, the present inventionprovides a large number of advantages over previously described systems.For example, by providing multi-layered devices and systems, the presentinvention substantially expands the amount of parallelization that canbe achieved using microfluidic systems. Furthermore, by taking advantageof both surfaces of planar substrates, the present invention alsopermits the optimal use of substrate materials, allowing furtherminiaturization of fluidic processes, as well as providing costadvantages in terms of substrate conservation. Finally, in the case ofsome types of microfluidic systems, such as electrokinetic basedmicrofluidic systems, these multi-layer devices permit the simultaneoususe of individual electrode interfaces on multiple layers of the device,thereby simplifying electrical control of such devices.

[0020] I. Multi-layer Devices

[0021] Generally, the present invention provides multi-layermicrofluidic systems, by providing additional substrate layers, e.g.,third, fourth, fifth and more substrate layers, mated with the typicallydescribed first and second layers. Microfabricated elements, e.g.,grooves, wells and the like, are manufactured into the surfaces betweenthe various substrate layers. These microfabricated elements define thevarious microfluidic aspects or structures of the overall device, e.g.,channels, chambers and the like. In preferred aspects, a separatemicroscale channel network is provided between each of the substratelayers.

[0022] As used herein, the term “microscale” or “microfabricated”generally refers to structural elements or features of a device whichhave at least one fabricated dimension in the range of from about 0.1 μmto about 500 μm. Thus, a device referred to as being microfabricated ormicroscale will include at least one structural element or featurehaving such a dimension. When used to describe a fluidic element, suchas a passage, chamber or conduit, the terms “microscale,”“microfabricated” or “microfluidic” generally refer to one or more fluidpassages, chambers or conduits which have at least one internalcross-sectional dimension, e.g., depth, width, length, diameter, etc.,that is less than 500 μm, and typically between about 0.1 μm and about500 μm. In the devices of the present invention, the microscale channelsor chambers preferably have at least one cross-sectional dimensionbetween about 0.1 μm and 200 μm, more preferably between about 0.1 μmand 100 μm, and often between about 0.1 μm and 20 μm. Accordingly, themicrofluidic devices or systems prepared in accordance with the presentinvention typically include at least one microscale channel, usually atleast two intersecting microscale channels, and often, three or moreintersecting channels disposed within a single body structure. Channelintersections may exist in a number of formats, including crossintersections, “T” intersections, or any number of other structureswhereby two channels are in fluid communication.

[0023] The body structure of the microfluidic devices described hereintypically comprises an aggregation of more than two separate substratelayers which when appropriately mated or joined together, form themicrofluidic device of the invention, e.g., containing the multiplechannel networks described herein. Typically, the microfluidic devicesdescribed herein will comprise at least three substrate layers,including a bottom substrate layer, a middle substrate layer and a topsubstrate layer. In some aspects the microfluidic devices of the presentinvention will include more than four, five, six, seven eight or moresubstrate layers, depending upon the nature of the operation for whichthe device is to be used.

[0024] As used herein, the terms “substrate” or “substrate layer” areused interchangeably to refer to solid planar substrates having firstand second opposing, or substantially parallel, planar surfaces. Avariety of substrate materials may be employed as the various layers ofthe device. Typically, because the devices are microfabricated,substrate materials will be selected based upon their compatibility withknown microfabrication techniques, e.g., photolithography, wet chemicaletching, laser ablation, air abrasion techniques, injection molding,embossing, and other techniques. The substrate materials are alsogenerally selected for their compatibility with the full range ofconditions to which the microfluidic devices may be exposed, includingextremes of pH, temperature, salt concentration, and application ofelectric fields. Substrates are also generally selected for theirelectrokinetic properties, e.g., surface potential, thermal and opticalproperties, e.g., transparency etc. Accordingly, in some preferredaspects, the substrate material may include materials normallyassociated with the semiconductor industry in which suchmicrofabrication techniques are regularly employed, including, e.g.,silica based substrates, such as glass, quartz, silicon or polysilicon,as well as other substrate materials, such as gallium arsenide and thelike. In the case of semiconductive materials, it will often bedesirable to provide an insulating coating or layer, e.g., siliconoxide, over the substrate material, and particularly in thoseapplications where electric fields are to be applied to the device orits contents.

[0025] In additional preferred aspects, the substrate materials willcomprise polymeric materials, e.g., plastics, such aspolymethylmethacrylate (PMMA), polycarbonate, polytetrafluoroethylene(TEFLON™), polyvinylchloride (PVC), polydimethylsiloxane (PDMS),polysulfone, and the like. Such polymeric substrates are readilymanufactured using available microfabrication techniques, as describedabove, or from microfabricated masters, using well known moldingtechniques, such as injection molding, embossing or stamping, or bypolymerizing the polymeric precursor material within the mold (See U.S.Pat. No. 5,512,131). Such polymeric substrate materials are preferredfor their ease of manufacture, low cost and disposability, as well astheir general inertness to most extreme reaction conditions. Again,these polymeric materials may include treated surfaces, e.g.,derivatized or coated surfaces, to enhance their utility in themicrofluidic system, e.g., provide enhanced fluid direction, e.g., asdescribed in U.S. Pat. No. 5,885,470 and which is incorporated herein byreference in its entirety for all purposes.

[0026] As noted above, the various substrate layers of the microfluidicdevices are mated or bonded together to form the microfluidic elementsof the device. Bonding of substrate layers is generally carried outunder any of a number of methods or conditions known in the art.Conditions under which substrates may be bonded together are generallywidely understood, and such bonding of substrates is generally carriedout by any of a number of methods, which may vary depending upon thenature of the substrate materials used. For example, thermal bonding ofsubstrates may be applied to a number of substrate materials, including,e.g., glass or silica based substrates, as well as polymer basedsubstrates. Such thermal bonding typically comprises mating together thesubstrates that are to be bonded, under conditions of elevatedtemperature and, in some cases, application of external pressure. Theprecise temperatures and pressures will generally vary depending uponthe nature of the substrate materials used.

[0027] For example, for silica-based substrate materials, i.e., glass(borosilicate glass, Pyrex™, soda lime glass, etc.), quartz, and thelike, thermal bonding of substrates is typically carried out by pressingthe substrates together at temperatures ranging from about 500° C. toabout 1400° C., and preferably, from about 500° C. to about 1200° C. Forexample, soda lime glass is typically bonded at temperatures around 550°C., whereas borosilicate glass typically is thermally bonded at or near800° C. Quartz substrates, on the other hand, are typically thermallybonded at temperatures at or near 1200° C. These bonding temperaturesare typically achieved by placing the substrates to be bonded into hightemperature annealing ovens. These ovens are generally commerciallyavailable from, e.g., Fischer Scientific, Inc., and LabLine, Inc.

[0028] Polymeric substrates that are thermally bonded on the other hand,will typically utilize lower temperatures and/or pressures thansilica-based substrates, in order to prevent excessive melting of thesubstrates and/or distortion, e.g., flattening of the interior portionof the device, i.e., channels or chambers. Generally, such elevatedtemperatures for bonding polymeric substrates will vary from about 80°C. to about 200° C., depending upon the polymeric material used, andwill preferably be between about 90° C. and 150° C. Because of thesignificantly reduced temperatures required for bonding polymericsubstrates, such bonding may typically be carried out without the needfor high temperature ovens, as used in the bonding of silica-basedsubstrates.

[0029] Adhesives may also be used to bond substrates together accordingto well known methods, which typically comprise applying a layer ofadhesive between the substrates that are to be bonded and pressing themtogether until the adhesive sets. A variety of adhesives may be used inaccordance with these methods, including, e.g., UV curable adhesives,that are commercially available. Alternative methods may also be used tobond substrates together in accordance with the present invention,including e.g., acoustic or ultrasonic welding, RF welding and/orsolvent welding of polymeric parts.

[0030] As used herein, the term “channel network” refers to one or moremicroscale channels that are disposed between two substrates. Inpreferred aspects, such channel networks include at least two microscalechannels, and preferably, at least two intersecting microscale channels.The intersection of channels can include channels which intersect andcross, e.g., at “four-way intersections, as well as a channelintersection wherein one channel intersects and terminates in anotherchannel, e.g., at a “T” or “three-way” intersection. In many aspects,the individual channel networks will preferably include at least threeintersecting channels, in some aspects, greater than four intersectingchannels and often greater than five, six or even eight intersectingchannels.

[0031] Typically included within a given channel network are channels inwhich the desired analysis is to take place, or “analysis channels.”Also, typically included are channels for delivering reagents, buffers,diluents, sample material and the like to the analysis channels.Analysis channels optionally include separation matrices disposedwithin, for separating/fractionating materials transported down thelength of these channels, for analysis, i.e., size or charged basedfractionation of materials, e.g., nucleic acids, proteins etc. Suitableseparation matrices include, e.g., GeneScan™ polymers (PerkinElmer-Applied Biosystems Division, Foster City, Calif.). Alternatively,analysis channels are devoid of any separation matrix, and instead,merely provide a channel within which an interaction, reaction etc.,takes place. Examples of microfluidic devices incorporating suchanalysis channels are described in, e.g., Published PCT Application No.WO 98/00231, and U.S. Pat. No. 5,976,336, each of which is herebyincorporated herein by reference in its entirety for all purposes.

[0032] One of the advantages of the present invention is to providemicrofluidic devices that make optimal use of the substrate materialfrom which they are fabricated. As such, in preferred aspects, thechannel networks used in the devices of the present invention willtypically exist in a limited space. Specifically, the channel networkswill typically be entirely incorporated in a substrate area that is lessthan 3 cm². In particularly preferred aspects, the entire channelnetwork will be incorporated in an area of less than 1 cm². Often, theentire channel network will be incorporated in less than 0.5 cm². Inorder to ensure that the channel networks are incorporated within thepreferred range of areas, any given straight portion of any channel ofthe device will typically have a length that is less than 1 cm,preferably, less than 0.8 cm, and in many cases, less than 0.5 cm.Specific channels may have overall lengths far greater than theselengths where those channels include serpentine or other turning or morecomplex geometries.

[0033] An example of a multi-layer microfluidic device according to thepresent invention is schematically illustrated in FIG. 1. FIG. 1Aillustrates the three-layer construction of the device from aperspective view. FIG. 1B illustrates a perspective view of an assembleddevice, e.g., where the layers are mated together. FIG. 1C illustrates aside view of the assembled device. As shown, the device 100 includesmultiple substrate layers, such as bottom substrate 102, middlesubstrate 104 and top substrate 106. Bottom substrate 102 includes a topsurface 112, which is mated with the bottom surface 114 of the middlesubstrate 104. The top surface 115 of the middle substrate 104 islikewise mated with the bottom surface 116 of the top substrate 106. Afirst channel network 122 is fabricated into the top surface 112 of thebottom substrate 102, as a series of grooves. A similar series ofgrooves is fabricated into the top surface 115 of the middle substrate104, to form a second channel network 124. Upon mating the top surfaceof the bottom substrate with the bottom surface of the middle substrate,these grooves form the channels of the device. Alternatively, thechannel network 122 is optionally fabricated onto the bottom surface ofmiddle substrate 104. Upon mating with bottom substrate 102, thechannels of the device are formed. This alternate method provides forcost savings where materials for the substrate incorporating the channelnetworks are substantially more costly than those substrates used ascover layers. Further, alignment of channel networks on different layersof the device is made more simple by their fabrication on a singlesubstrate.

[0034] A plurality of ports is also provided through the substratelayers of the device, to provide access to the channel networks. In thedevices of the present invention, each of the channel networks of thedevice is in fluid communication with at least two ports or reservoirsdisposed in the body of the device. In preferred aspects, at least oneport is common to, i.e., in simultaneous fluid communication with, twoor more channel networks, while at least one port is typically specificto a single channel network.

[0035] In order to be common to multiple channel networks in themulti-layered devices of the invention, a port must traverse multiplesubstrate layers. An example of this port structure is illustrated inFIGS. 1A-C. For example, ports 126 and 128 are shown in FIGS. 1A and 1C,as apertures disposed through upper substrate 106 and middle substrate104, providing access to and fluid communication with both channelnetwork 124 and channel network 122. Meanwhile, port 132 is showndisposed through only the top substrate 106, whereupon it is in fluidcommunication only with channel network 124. Port 130, on the other handis disposed through top substrate 106 and middle substrate 104, butoriented at a position in the substrate such that it only providesaccess to, and is in fluid communication with only channel network 122.This is a result of channel network 124 not extending to andintersecting port 130. A schematic illustration of this type of portstructure is shown in FIG. 1C, from a side view.

[0036] In some instances, the ports of the device, common or otherwise,may be disposed through all of the layers of the device. For example,with reference to the devices illustrated in FIGS. 1A, 1B and 1C, ports126, 128 and 130 can optionally be disposed through bottom substrate102, in addition to being disposed through middle and top substrates 104and 106, respectively. Such port structures are particularly usefulwhere extremely small fluid volumes are to be introduced into device,requiring small diameter ports. Specifically, small diameter portshaving a closed end can result in the trapping of bubbles below a volumeof fluid that is placed into the port, due to surface tension effects ofthe fluid. However, by providing a port having an open bottom, there isprovided an escape route for the bubbles. Additionally, the fluidintroduced into the port is maintained within the port by virtue of thecapillary forces exerted by the walls of the port. This type of portstructure is described in substantial detail in commonly owned U.S. Pat.No. 6,090,251, and incorporated herein by reference.

[0037] In addition to providing fluid access and/or storage for thechannel networks of the device, the ports of the device also typicallyprovide access for the material direction and transport systems, whichmove and direct materials and/or fluids through the various channelnetworks of the device. Such fluid direction and transport systemstypically include, e.g., micromechanical systems employing micropumpsand microvalves, pressure based and/or pneumatic systems which move anddirect materials by application of pressure differentials acrosschannels, electrokinetic material transport systems which move materialsby applying electric fields across channel lengths, and the like.

[0038] Pressure based material transport systems typically operate byapplying a pressure differentials from one end of a channel throughwhich material movement is desired, to the other end of the channel. Byapplying appropriate pressures across intersecting channels, one cancontrol material direction through the intersections of channels, and inand out of chambers (see, e.g., Published PCT Application No WO97/02357. Typically, controlled pressures are applied to the ports ofthe microfluidic device, which ports are in communication with thechannel networks.

[0039] In particularly preferred aspects, the devices and systems of thepresent invention utilize electrokinetic material transport anddirection systems. “Electrokinetic material transport and directionsystems,” as described herein, include systems which transport anddirect materials within an interconnected channel and/or chambercontaining structure, through the application of electrical fields tothe materials. Application of this electric field causes materialmovement through and among the channels and/or chambers, i.e., cationswill move toward the negative electrode, while anions will move towardthe positive electrode.

[0040] Such electrokinetic material transport and direction systemsinclude those systems that rely upon the electrophoretic mobility ofcharged species within the electric field applied to the structure. Suchsystems are more particularly referred to as electrophoretic materialtransport systems. In particularly preferred aspects, however, theelectrokinetic material direction and transport systems rely upon theelectroosmotic flow of fluid and material within a channel or chamberstructure which results from the application of an electric field acrosssuch structures. In brief, when a fluid is placed into a channel whichhas a surface bearing charged functional groups, e.g., hydroxyl groupsin etched glass channels or glass microcapillaries, those groups canionize. In the case of hydroxyl functional groups, this ionization,e.g., at neutral pH, results in the release of protons from the surfaceand into the fluid, creating a concentration of protons at near thefluid/surface interface, or a positively charged sheath surrounding thebulk fluid in the channel. Application of a voltage gradient across thelength of the channel will cause the proton sheath to move in thedirection of the voltage drop, i.e., toward the negative electrode.

[0041] “Controlled electrokinetic material transport and direction,” asused herein, refers to electrokinetic systems as described above, whichemploy active control of the voltages applied at multiple, i.e., morethan two, electrodes. Rephrased, such controlled electrokinetic systemsconcomitantly regulate voltage gradients applied across at least twointersecting channels. Controlled electrokinetic material transport isdescribed in Published PCT Application No. WO 96/04547 to Ramsey, whichis incorporated herein by reference in its entirety for all purposes. Inparticular, the preferred microfluidic devices and systems describedherein, include a body structure which includes at least twointersecting channels or fluid conduits, e.g., interconnected, enclosedchambers, which channels include at least three unintersected termini.The intersection of two channels refers to a point at which two or morechannels are in fluid communication with each other, and encompasses “T”intersections, cross intersections, “wagon wheel” intersections ofmultiple channels, or any other channel geometry where two or morechannels are in such fluid communication. An unintersected terminus of achannel is a point at which a channel terminates not as a result of thatchannel's intersection with another channel, e.g., a “T” intersection.In preferred aspects, the devices will include at least threeintersecting channels having at least four unintersected termini. In abasic cross channel structure, where a single horizontal channel isintersected and crossed by a single vertical channel, controlledelectrokinetic material transport operates to controllably directmaterial flow through the intersection, by providing constraining flowsfrom the other channels at the intersection. For example, assuming onewas desirous of transporting a first material through the horizontalchannel, e.g., from left to right, across the intersection with thevertical channel. Simple electrokinetic flow of this material across theintersection could be accomplished by applying a voltage gradient acrossthe length of the horizontal channel, i.e., applying a first voltage tothe left terminus of this channel, and a second, lower voltage to theright terminus of this channel, or by grounding that terminus. However,this type of material flow through the intersection results in asubstantial amount of diffusion at the intersection, resulting from boththe natural diffusive properties of the material being transported inthe medium used, as well as convective effects at the intersection.

[0042] In controlled electrokinetic material transport, the materialbeing transported across the intersection is constrained by low levelflow from the side channels, e.g., the top and bottom channels. This isaccomplished by applying a slight voltage gradient along the path ofmaterial flow, e.g., from the top or bottom termini of the verticalchannel, toward the right terminus. The result is a “pinching” of thematerial flow at the intersection, which prevents the diffusion of thematerial into the vertical channel. The pinched volume of material atthe intersection may then be injected into the vertical channel byapplying a voltage gradient across the length of the vertical channel,i.e., from the top terminus to the bottom terminus. In order to avoidany bleeding over of material from the horizontal channel during thisinjection, a low level of flow is directed back into the side channels,resulting in a “pull back” of the material from the intersection.

[0043] In addition to pinched injection schemes, controlledelectrokinetic material transport is readily utilized to create virtualvalves which include no mechanical or moving parts. Specifically, withreference to the cross intersection described above, flow of materialfrom one channel segment to another, e.g., the left arm to the right armof the horizontal channel, can be efficiently regulated, stopped andreinitiated, by a controlled flow from the vertical channel, e.g., fromthe bottom arm to the top arm of the vertical channel. Specifically, inthe ‘off’ mode, the material is transported from the left arm, throughthe intersection and into the top arm by applying a voltage gradientacross the left and top termini. A constraining flow is directed fromthe bottom arm to the top arm by applying a similar voltage gradientalong this path (from the bottom terminus to the top terminus). Meteredamounts of material are then dispensed from the left arm into the rightarm of the horizontal channel by switching the applied voltage gradientfrom left to top, to left to right. The amount of time and the voltagegradient applied dictates the amount of material that will be dispensedin this manner.

[0044] Although described for the purposes of illustration with respectto a four way, cross intersection, these controlled electrokineticmaterial transport systems can be readily adapted for more complexinterconnected channel networks, e.g., arrays of interconnected parallelchannels.

[0045] The above described microfluidic devices provide enhanced abilityto perform parallel analyses on a single material placed into a singlemicrofluidic device, i.e., serial or single to parallel conversion.However, the multi-layer microfluidic devices of the present inventionalso permit greater freedom in constructing channel networks.Specifically, such multi-layer devices include channels or channelnetworks that bridge other channels or channel networks, by detouringfrom one layer of the device to the other. Thus, in addition toproviding multi-layer microfluidic devices wherein channel networks ondifferent layers are in fluid communication via common ports, thepresent invention also provides devices that include fluid passagesdisposed through substrates, in order to provide fluid communicationbetween channels or channel networks that are disposed on differentlayers of the device. By permitting such cross-over channel structures,the present invention provides greater freedom in designing channelnetworks, as well as greater channel density per unit area of substrate.An example of a cross over channel structure in a multi-layer device isshown in FIG. 3.

[0046]FIGS. 3A and 3B illustrate a portion of a multi-layer microfluidicdevice. The device is again fabricated from a bottom substrate 102,middle substrate 104 and top substrate 106. The device portion isillustrated from a side view (FIG. 3A) and a top view (FIG. 3B). Thedevice includes channels 302, 304, 306 and 308 (channel 308 is shownfrom the end view) fabricated into the upper surface of the middlesubstrate 104. Channel 302 is in fluid communication with channel 304via channel 306 which, as shown, is fabricated into the bottom surfaceof the middle substrate 104. Each of channels 302 and 304 communicateswith channel 306 via a passage 310 fabricated through the middlesubstrate layer. These fluid passages 310 are generally fabricated usingthe same methods used in producing the port structures described withreference to FIG. 1. For example, microscale drills, air abrasion, laserablation and like methods are readily applicable to fabricating thesepassages through the substrates. Further, for thinner substratematerials, chemical etching techniques are also useful in fabricatingthese passages.

[0047] Because these passages are intended for fluid transport, ratherthan fluid introduction, i.e., as in the case of the ports describedwith reference to FIG. 1, they will generally have a substantiallysmaller diameter. Specifically, in the microfluidic systems describedherein, it is generally advantageous to minimize any dead volumes withinthe device to avoid negating any advantages otherwise gained in lowvolume or high throughput processing. For example, the ports describedabove will generally range from about 0.5 mm to about 10 mm, andpreferably from about 11 mm to about 5 mm, the fluid passages disposedthrough the substrates on the other hand, will typically range fromabout 30 μm to about 500 μm in diameter, and preferably will befabricated to a similar cross section as the channels with which thepassages communicate. Again, as with the devices illustrated in FIG. 1,the device partially shown in FIG. 3 also typically includes ports incommunication with the channels of the device (ports not shown).Material direction and transport is also preferably accomplished usingcontrolled electrokinetic transport methods, as described above.

[0048] II. Instrumentation

[0049] The systems described herein generally include microfluidicdevices, as described above, in conjunction with additionalinstrumentation. The additional instrumentation typically includes powersupplies and controllers, for controlling fluid transport and directionwithin the devices, as described in greater detail below. Theinstrumentation also typically includes detection instrumentation fordetecting or sensing results of the operations performed by the system.Further, the instrumentation often includes processor instrumentation,e.g., computers etc., for instructing the controlling instrumentation inaccordance with preprogrammed instructions, receiving data from thedetection instrumentation, and for analyzing, storing and interpretingthe data, and providing the data and interpretations in a readilyaccessible reporting format.

[0050] A variety of controlling instrumentation may be utilized inconjunction with the microfluidic devices described above, forcontrolling the transport and direction of fluids and/or materialswithin the devices of the present invention, in accordance with thesystems described above. Typically, such controllers incorporate thedriving system for the material transport system, e.g., electrical powersources, pressure sources etc. The controller also typically includes anappropriate interface component, for interfacing the microfluidic devicewith he transport driving system, e.g., power source, pressure source,etc. As noted above, the systems described herein preferably utilizeelectrokinetic material direction and transport systems. As such, thecontroller systems for use in conjunction with the microfluidic devicestypically include an electrical power supply and circuitry forconcurrently delivering appropriate voltages to a plurality ofelectrodes that are placed in electrical contact with the fluidscontained within the microfluidic devices via the ports described above.Examples of particularly preferred electrical controllers include thosedescribed in, e.g., Published PCT Application No. WO 98/00707, thedisclosure of which is hereby incorporated herein by reference in theirentirety for all purposes. In brief, the controller uses controlledelectric current or power to control material transport the microfluidicsystem. The electrical current flow at a given electrode is directlyrelated to the ionic flow along the channel(s) connecting the reservoirin which the electrode is placed. This is in contrast to the requirementof determining voltages at various nodes along the channel in a voltagecontrol system. Thus the voltages at the electrodes of the microfluidicsystem are set responsive to the electric currents flowing through thevarious electrodes of the system. This current control is lesssusceptible to dimensional variations in the process of creating themicrofluidic system in the device itself. Current control permits fareasier operations for pumping, valving, dispensing, mixing andconcentrating subject materials and buffer fluids in a complexmicrofluidic system.

[0051] In the microfluidic systems described herein, a variety ofdetection methods and systems may be employed, depending upon thespecific operation that is being performed by the system. Often, amicrofluidic system will employ multiple different detection systems formonitoring the output of the system. Examples of detection systemsinclude optical sensors, temperature sensors, electrochemical sensors,pressure sensors, pH sensors, conductivity sensors, and the like. Eachof these types of sensors is readily incorporated into the microfluidicsystems described herein. In these systems, such detectors are placedeither within or adjacent to the microfluidic device or one or morechannels, chambers or conduits of the device, such that the detector iswithin sensory communication with the device, channel, or chamber.

[0052] The phrase “within sensory communication” of a particular regionor element, as used herein, generally refers to the placement of thedetector in a position such that the detector is capable of detectingthe property of the microfluidic device, a portion of the microfluidicdevice, or the contents of a portion of the microfluidic device, forwhich that detector was intended. For example, a pH sensor placed insensory communication with a microscale channel is capable ofdetermining the pH of a fluid disposed in that channel. Similarly, atemperature sensor placed in sensory communication with the body of amicrofluidic device is capable of determining the temperature of thedevice itself.

[0053] Particularly preferred detection systems include opticaldetection systems for detecting an optical property of a material withinthe channels and/or chambers of the microfluidic devices that areincorporated into the microfluidic systems described herein. Suchoptical detection systems are typically placed adjacent a microscalechannel of a microfluidic device, and are in sensory communication withthe channel via an optical detection window that is disposed across thechannel or chamber of the device. Optical detection systems includesystems that are capable of measuring the light emitted from materialwithin the channel, the transmissivity or absorbance of the material, aswell as the materials spectral characteristics. In preferred aspects,the detector measures an amount of light emitted from the material, suchas a fluorescent or chemiluminescent material.

[0054] The detector may exist as a separate unit, but is preferablyintegrated with the controller system, into a single instrument.Integration of these functions into a single unit facilitates connectionof these instruments with the computer (described below), by permittingthe use of few or a single communication port(s) for transmittinginformation between the controller, the detector and the computer.Additionally, the detector and/or the device will typically include aninterface element for placing the detector in sensory communication witha channel of the device. In the case of potentiometric detectionsystems, such interface components typically include electricalcouplings on the detector and device, for connecting the sensor portionof the device to the detector portion of the controller. Alternatively,the interface component can include thin regions of the device's bodystructure which when the device is mounted on the controller, place thedetector in sensory communication with the channels of the device, e.g.,to measure temperature or some optical property within a channel.

[0055] In preferred aspects, the devices of the present inventiontypically include one or more detection windows, for detecting theprogress and/or results of a particular operation being performed withinthe device. As used herein, the term “detection window” refers to aregion on the body of a microfluidic device that is capable oftransmitting a detectable signal from one or more microfluidic channelsof the device to the exterior of the device, where it can be readilydetected, e.g., by a detector disposed adjacent the detection window.FIG. 1B illustrates the orientation of these detection windows 136 and138, on the body of the device 100 which incorporates two separatechannel networks, and consequently, two detection windows 136 and 138.Although it is preferred to have separate detection windows for separatechannel structures, it is not required. For example, a single detectionwindow is used where two channel networks overlay each other, or are inclose proximity to each other, but produce readily distinguishablesignals, such as different signal types, e.g., light vs. radiation,emitted fluorescence at different wavelengths, and the like. Inpreferred aspects, the detection windows are optical detection widows,permitting the transmission of an optical signal from one or morechannels or channel networks to the exterior of the device, fordetection by an appropriately positioned optical detector.

[0056] In the case of multi-layer devices, some of the channel networksmay include multiple substrate layers between the channel and thedetector. Although selection of highly pure substrate materials canreduce the amount of interference caused by these thicker substrates,e.g., autofluorescence and the like, as well as increase lightcollection efficiencies, it is generally desirable to minimize thenumber of substrate layers or amount of substrate material between thechannel network and the detector, at the detection window. Generally,this is accomplished by providing windows or apertures through the uppersubstrate layers so as to minimize the number of substrate layersthrough which the signal must pass in order to reach the detector. Oneexample of this detection window structure is shown in FIG. 1C, whichillustrates a detection window for the first channel network 122,including an aperture 134 disposed entirely through, or optionallypartially through the top substrate 106, whereby the signal from channelnetwork 122 is only required to pass through the middle substrate 104before it reaches the detector. An alternative structure is illustratedin FIG. 2, wherein the upper substrate layers are staggered to providean unobstructed detection window for the channel networks disposedbetween the lower substrate layers.

[0057] In the case of optical detection, the detection system willtypically include collection optics for gathering a light based signaltransmitted through the detection window, and transmitting that signalto an appropriate light detector. Microscope objectives of varyingpower, field diameter, and focal length may be readily utilized as atleast a portion of this optical train. The light detectors may bephotodiodes, avalanche photodiodes, photomultiplier tubes, diode arrays,or in some cases, imaging systems, such as charged coupled devices(CCDs) and the like. In preferred aspects, photodiodes are utilized, atleast in part, as the light detectors. The detection system is typicallycoupled to the computer (described in greater detail below), via anAD/DA converter, for transmitting detected light data to the computerfor analysis, storage and data manipulation.

[0058] In the case of fluorescent materials, the detector will typicallyinclude a light source which produces light at an appropriate wavelengthfor activating the fluorescent material, as well as optics for directingthe light source through the detection window to the material containedin the channel or chamber. The light source may be any number of lightsources that provides the appropriate wavelength, including lasers,laser diodes and LEDs. Other light sources may be required for otherdetection systems. For example, broad band light sources are typicallyused in light scattering/transmissivity detection schemes, and the like.Typically, light selection parameters are well known to those of skillin the art.

[0059] As noted above, either or both of the controller system and/orthe detection system are coupled to an appropriately programmedprocessor or computer which functions to instruct the operation of theseinstruments in accordance with preprogrammed or user input instructions,receive data and information from these instruments, and interpret,manipulate and report this information to the user. As such, thecomputer is typically appropriately coupled to one or both of theseinstruments (e.g., including an AD/DA converter as needed).

[0060] The computer typically includes appropriate software forreceiving user instructions, either in the form of user input into a setparameter fields, e.g., in a GUI (graphical user interface), or in theform of preprogrammed instructions, e.g., preprogrammed for a variety ofdifferent specific operations. The software then converts theseinstructions to appropriate language for instructing the operation ofthe fluid direction and transport controller to carry out the desiredoperation. The computer then receives the data from the one or moresensors/detectors included within the system, and interprets the data,either provides it in a user understood format, or uses that data toinitiate further controller instructions, in accordance with theprogramming, e.g., such as in monitoring and control of flow rates,temperatures, applied voltages, and the like.

[0061] III. Exemplary Applications

[0062] The multi-layer microfluidic devices described herein, have anextremely wide range of applications, including high throughputscreening of chemical libraries in screening for lead compounds in drugdiscovery, nucleic acid analysis for genomic research, genetic testingand identification, and a myriad of other analytical operations.

[0063] One example of an application in which these multi-layer systemsare particularly useful is in the performance of nucleic acid sequencingoperations. For example, the multi-layer systems can be used to performsimultaneous sample preparation, separations and analysis for each offour separate sample treatments under either the Sanger or Maxam-Gilbertsequencing methods.

[0064] In particular, with reference to the Sanger dideoxynucleotidesequencing process and FIG. 4, a single microfluidic device 400 includesfour separate channel networks 410, 420, 430 and 440, each disposed in aseparate layer of the device. Each of the channel networks includes acorresponding sample channel 411, 421, 431 and 441 in communication witha common sample port 402 and waste port 404. The sample containing thetarget nucleic acid that is to be sequenced, is deposited in the sampleport 402, along with an effective concentration of DNA polymerase andprimer sequence.

[0065] Each of the sample channels 411, 421, 431, and 441, isintersected by a corresponding second channel 412, 422, 432 and 442.Each of these channels leads to and is in fluid communication with aseparate one of four different ports 413, 423, 433 and 443. In each ofthese ports is deposited a mixture of the four deoxynucleosidetriphosphates (dNTPs:A; T; G; and C), as well as one of the fourdideoxynucleoside triphosphates (ddNTPs: ddA, ddC; ddG; ddT), and otherappropriate reagents, e.g., Mg++.

[0066] The sample is transported, simultaneously, along each of the foursample channels 411, 421, 431 and 441. The cocktail of dNTPs and theddNTP is introduced into the channel at the intersection of the samplechannel and the NTP channel, whereupon it mixes with the sample andbegins extending the primer sequences along the length of the targetsequence. For polymerase reactions requiring longer reaction times,e.g., for longer sequences, the sample channel can be elongated, oralternatively, widened in one region to provide a mixing/reactionchamber, to complete the polymerase reaction prior to separation of thereaction products. Similarly, flow rates are optionally slowed to allowmore contact/mixing time between reagents.

[0067] The inclusion of the dideoxynucleotide results in the productionof a nested set of nucleic acid polymerase products that terminates atthe point the ddNTP was incorporated, indicating the complementary basein the target sequence, for that position. The mixture of reactionproducts is then transported across the intersection of the samplechannel (411, 421, 431 or 441) and the corresponding separation channel(414, 424, 434, or 444, respectively) toward waste ports 415, 425, 435and 445, respectively. The plug of reaction products present in theintersection is then electrophoresed down separation channel, separatingthe reaction products based upon size. Comparative analysis of thereaction product sizes in each different channel network permits thedetermination of the sequence. For additional complexity, differentcolor detectable labels are incorporated in each of the separatereactions, to facilitate differentiation of signals from each channelnetwork. However, single color detection is readily employed byproviding for separate detection of separated reaction products in eachof the four different reactions/channel networks, e.g., using detectionwindows disposed over each of the separation channels 415, 425, 435 or445.

[0068] A similar device construction is used for performing degradativesequencing of nucleic acids, e.g., Maxam-Gilbert. In particular, insteadof providing a different cocktail of dNTPs and ddNTP in each of the fourports 413, 423, 433 and 443, one merely deposits a different cleavagereagent, e.g., hydrazine or piperidine, or subjects the particularchannel network to appropriate cleavage conditions, e.g., methylationand acid or base cleavage. See, e.g., Biochemistry by Lubert Stryer,(W.H. Freeman and Co.).

[0069] All publications and patent applications are herein incorporatedby reference to the same extent as if each individual publication orpatent application was specifically and individually indicated to beincorporated by reference. Although the present invention has beendescribed in some detail by way of illustration and example for purposesof clarity and understanding, it will be apparent that certain changesand modifications may be practiced within the scope of the appendedclaims.

What is claimed is:
 1. A microfluidic device comprising: a bodystructure having at least first, second and third substrate layers, thesecond substrate layer disposed on top of the first substrate layer andthe third substrate layer disposed on top of the second substrate layer;at least first, second and third ports disposed in the body structure;and at least first and second microscale channel networks, the firstchannel network disposed between the first and second substrate layers,and being in fluid communication with the first and second ports, butnot the third port, and the second channel network disposed between thesecond and third layers, and being in fluid communication with the firstand third ports, but not the second port.